Method and nucleic acids for the analysis of colon cancer

ABSTRACT

The present invention relates to chemically modified genomic sequences, oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genomic DNA, as well as to methods for ascertaining genetic and/or epigenetic parameters of genes for use in the characterisation, grading, staging, and/or diagnosis of colon cancer, or the predisposition to colon cancer.

FIELD OF THE INVENTION

The levels of observation that have been studied by the methodologicaldevelopments of recent years in molecular biology, are the genesthemselves, the translation of these genes into RNA, and the resultingproteins. The question of which gene is switched on at which point inthe course of the development of an individual, and how the activationand inhibition of specific genes in specific cells and tissues arecontrolled is correlatable to the degree and character of themethylation of the genes or of the genome. In this respect, pathogenicconditions may manifest themselves in a changed methylation pattern ofindividual genes or of the genome.

The present invention relates to nucleic acids, oligonucleotides,PNA-oligomers, and to a method for the characterisation, grading,staging, treatment and/or diagnosis of colon cancer, or thepredisposition to colon cancer, by analysis of the genetic and/orepigenetic parameters of genomic DNA and, in particular, with thecytosine methylation status thereof.

PRIOR ART

Colon cancer is the second most common cause of cancer death in theUnited States. It describes any cancer in the colon (large intestine),from the beginning of the colon (cecum) to the end of the colon(rectum). Colon cancer is a malignant tumor in the lining of the largeintestine. It starts with a single cell that mutates and grows into avisible polyp. If a polyp is allowed to remain in the colon it can growinto a cancerous tumor that can invade other organs. The mechanismbehind the progression to malignancy are not comletely understood,however most polyps take 3-7 years to become cancerous. Prevention ofcolon cancer means stopping this process by removing the polyp before itbecomes cancerous. Colon cancer represents an interaction between thegenome of the colorectal epithelial cell and the host environment. Bothfactors are essential for the development of tumors. Colon cancers canbe differentiated into nonhereditary types, which rarely occur beforeage 40 and hereditary colon cancers which often occur in younger people.

Human colon cancers undergo a multistage carcinogenesis pathway fromadenomatous polyps to carcinoma. A number of genetic events have beencharacterized and include alterations in “tumor suppressor” andsusceptibility genes that normally encode for proteins regulating cellcycle progression and programmed cell death (Kinzler K W, Vogelstein B.Landscaping the cancer terrain. Science. 1998 May 15;280(5366):1036-7).Given the high incidence of colon cancer in the aging population andhigh mortality rates for advanced disease, new prevention strategies areneeded. After the diagnosis of cancer has been made it is important todetermine the extent or ‘stage’ of the cancer before deciding on thetreatment plan. Staging is a method of evaluating the progress of thecancer in a patient and defines the extent to which the cancer hasspread to other parts of the body. There are several systems forclassifying the extent or stage of cancer. One of the the two mostcommon systems is the Stage ‘I, II, III, IV’ system, which defines fourstages of cancer. Stage I represents early cancer, with a small tumorand no spread to the lymph nodes. In stages II and III, the tumor isprogressively more advanced, while stage IV refers to metastatic diseasethat has spread to other areas of the body. One very important point torealize about these staging systems is that they only provide roughestimates of the stage of disease and chances of survival. The numbersare just averages. They do not say anything about the outcome orprognosis of any one particular patient.

Genes which are associated with colon cancer include the following.

-   p16 (Dai C Y, Furth E E, Mick R, Koh J, Takayama T, Niitsu Y, Enders    G H. p16(INK4a) expression begins early in human colon neoplasia and    correlates inversely with markers of cell proliferation.    Gastroenterology. 2000 October; 119(4):929-42).-   p27 (Liu D F, Ferguson K, Cooper G S, Grady W M, Willis J. p27    cell-cycle inhibitor is inversely correlated with lymph node    metastases in right-sided colon cancer. J Clin Lab Anal.    1999;13(6):291-5).-   p53 (Arango D, Corner G A, Wadler S, Catalano P J, Augenlicht L H.    c-myc/p53 interaction determines sensitivity of human colon    carcinoma cells to 5-fluorouracil in vitro and in vivo. Cancer Res.    2001 Jun. 15;61(12):4910-5).-   cdc2 (Moragoda L, Jaszewski R, Majumdar A P. Curcumin induced    modulation of cell cycle and apoptosis in gastric and colon cancer    cells. Anticancer Res. 2001 March-April; 21(2A):873-8).-   PCNA (Zhang Y, Iwama T, Sugihara K. Histochemical study of apoptosis    and cell proliferation in hereditary intestinal diseases. J Med Dent    Sci. 1998 June;45(2):77-84).-   CEA (Vogel I, Francksen H, Soeth E, Henne-Bruns D, Kremer B, Juhl H.    The carcinoembryonic antigen and its prognostic impact on    immunocytologically detected intraperitoneal colorectal cancer    cells. Am J. Surg. 2001 February; 181(2):188-93).-   c-erbB2 (Fric P, Sovova V, Sloncova E, Lojda Z, Jirasek A, Cermak J.    Different expression of some molecular markers in sporadic cancer of    the left and right colon. Eur J Cancer Prev. 2000 August;    9(4):265-8).-   Estrogen receptor (Campbell-Thompson M, Lynch I J, Bhardwaj B.    Expression of estrogen receptor (ER) subtypes and ERbeta isoforms in    colon cancer. Cancer Res. 2001 Jan. 15;61(2):632-40).-   Progesterone receptor (Reich O, Regauer S, Urdl W, Lahousen M,    Winter R. Expression of oestrogen and progesterone receptors in    low-grade endometrial stromal sarcomas. Br J Cancer. 2000    March;82(5):1030-4) and myoglobin (Nakao A, Sakagami K, Uda M,    Mitsuoka S, Ito H. Carcinosarcoma of the colon: report of a case and    review of the literature. J Gastroenterol. 1998 April;33(2):276-9).

5-methylcytosine is the most frequent covalent base modification in theDNA of eukaryotic cells. It plays a role, for example, in the regulationof the transcription, in genetic imprinting, and in tumorigenesis.Therefore, the identification of 5-methylcytosine as a component ofgenetic information is of considerable interest. However,

5-methylcytosine positions cannot be identified by sequencing since5-methylcytosine has the same base pairing behavior as cytosine.Moreover, the epigenetic information carried by 5-methylcytosine iscompletely lost during PCR amplification.

A relatively new and currently the most frequently used method foranalyzing DNA for 5-methylcytosine is based upon the specific reactionof bisulfite with cytosine which, upon subsequent alkaline hydrolysis,is converted to uracil which corresponds to thymidine in its basepairing behavior. However, 5-methylcytosine remains unmodified underthese conditions. Consequently, the original DNA is converted in such amanner that methylcytosine, which originally-could not be distinguishedfrom cytosine by its hybridization behavior, can now be detected as theonly remaining cytosine using “normal” molecular biological techniques,for example, by amplification and hybridization or sequencing. All ofthese techniques are based on base pairing which can now be fullyexploited. In terms of sensitivity, the prior art is defined by a methodwhich encloses the DNA to be analyzed in an agarose matrix, thuspreventing the diffusion and renaturation of the DNA (bisulfite onlyreacts with single-stranded DNA), and which replaces all precipitationand purification steps with fast dialysis (Olek A, Oswald J, Walter J. Amodified and improved method for bisulphite based cytosine methylationanalysis. Nucleic Acids Res. 1996 Dec. 15;24(24):5064-6). Using thismethod, it is possible to analyze individual cells, which illustratesthe potential of the method. However, currently only individual regionsof a length of up to approximately 3000 base pairs are analyzed, aglobal analysis of cells for thousands of possible methylation events isnot possible. However, this method cannot reliably analyze very smallfragments from small sample quantities either. These are lost throughthe matrix in spite of the diffusion protection.

An overview of the further known methods of detecting 5-methylcytosinemay be gathered from the following review article: Rein, T.,DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998, 26, 2255.

To date, barring few exceptions (e.g., Zeschnigk M, Lich C, Buiting K,Doerfler W, Horsthemke B. A single-tube PCR test for the diagnosis ofAngelman and Prader-Willi syndrome based on allelic methylationdifferences at the SNRPN locus. Eur J Hum Genet. 1997March-April;5(2):94-8) the bisulfite technique is only used in research.Always, however, short, specific fragments of a known gene are amplifiedsubsequent to a bisulfite treatment and either completely sequenced(Olek A, Walter J. The pre-implantation ontogeny of the H19 methylationimprint. Nat Genet. 1997 November;17(3):275-6) or individual cytosinepositions are detected by a primer extension reaction (Gonzalgo M L,Jones P A. Rapid quantitation of methylation differences at specificsites using methylation-sensitive single nucleotide primer extension(Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15;25(12):2529-31, WO Patent9500669) or by enzymatic digestion (Xiong Z, Laird P W. COBRA: asensitive and quantitative DNA methylation assay. Nucleic Acids Res.1997 Jun. 15;25(12):2532-4). In addition, detection by hybridization hasalso been described (Olek et al., WO 99 28498).

Further publications dealing with the use of the bisulfite technique formethylation detection in individual genes are: Grigg G, Clark S.Sequencing 5-methylcytosine residues in genomic DNA. Bioessays. 1994June;16(6):431-6, 431; Zeschnigk M, Schmitz B, Dittrich B, Buiting K,Horsthemke B, Doerfler W. Imprinted segments in the human genome:different DNA methylation patterns in the Prader-Willi/Angelman syndromeregion as determined by the genomic sequencing method. Hum Mol Genet.1997 March;6(3):387-95; Feil R, Charlton J, Bird A P, Walter J, Reik W.Methylation analysis on individual chromosomes: improved protocol forbisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb.25;22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio M C, Dante R.Genomic sequencing indicates a correlation between DNA hypomethylationin the 5′ region of the pS2 gene and its expression in human breastcancer cell lines. Gene. 1995 May 19;157(1-2):261-4; WO 97/46705, WO95/15373 and WO 95/45560.

An overview of the Prior Art in oligomer array manufacturing can begathered from a special edition of Nature Genetics (Nature GeneticsSupplement, Volume 21, January 1999), published in January 1999, andfrom the literature cited therein.

Fluorescently labeled probes are often used for the scanning ofimmobilized DNA arrays. The simple attachment of Cy3 and Cy5 dyes to the5′-OH of the specific probe are particularly suitable for fluorescencelabels. The detection of the fluorescence of the hybridized probes maybe carried out, for example via a confocal microscope. Cy3 and Cy5 dyes,besides many others, are commercially available.

Matrix Assisted Laser Desorption Ionization Mass Spectrometry(MALDI-TOF) is a very efficient development for the analysis ofbiomolecules (Karas M, Hillenkamp F. Laser desorption ionization ofproteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988Oct. 15;60(20):2299-301). An analyte is embedded in a light-absorbingmatrix. The matrix is evaporated by a short laser pulse thustransporting the analyte molecule into the vapor phase in anunfragmented manner. The analyte is ionized by collisions with matrixmolecules. An applied voltage accelerates the ions into a field-freeflight tube. Due to their different masses, the ions are accelerated atdifferent rates. Smaller ions reach the detector sooner than biggerones.

MALDI-TOF spectrometry is excellently suited to the analysis of peptidesand proteins. The analysis of nucleic acids is somewhat more difficult(Gut I G, Beck S. DNA and Matrix Assisted Laser Desorption IonizationMass Spectrometry. Current Innovations and Future Trends. 1995, 1;147-57). The sensitivity to nucleic acids is approximately 100 timesworse than to peptides and decreases disproportionally with increasingfragment size. For nucleic acids having a multiply negatively chargedbackbone, the ionization process via the matrix is considerably lessefficient. In MALDI-TOF spectrometry, the selection of the matrix playsan eminently important role. For the desorption of peptides, severalvery efficient matrixes have been found which produce a very finecrystallization. There are now several responsive matrixes for DNA,however, the difference in sensitivity has not been reduced. Thedifference in sensitivity can be reduced by chemically modifying the DNAin such a manner that it becomes more similar to a peptide.Phosphorothioate nucleic acids in which the usual phosphates of thebackbone are substituted with thiophosphates can be converted into acharge-neutral DNA using simple alkylation chemistry (Gut I G, Beck S. Aprocedure for selective DNA alkylation and detection by massspectrometry. Nucleic Acids Res. 1995 Apr. 25;23(8):1367-73). Thecoupling of a charge tag to this modified DNA results in an increase insensitivity to the same level as that found for peptides. A furtheradvantage of charge tagging is the increased stability of the analysisagainst impurities which make the detection of unmodified substratesconsiderably more difficult.

Genomic DNA is obtained from DNA of cell, tissue or other test samplesusing standard methods. This standard methodology is found in referencessuch as Fritsch and Maniatis eds., Molecular Cloning: A LaboratoryManual, 1989.

DESCRIPTION OF THE INVENTION

The present invention discloses that atypical methylation in the genesestrogen receptor, p21, p27, p16, progesteron receptor, myoglobin, pcna,cdc2, c-erbB2, p53 and CEA, can be positively correlated with coloncarcinogenesis. This allows the detection of colon carcinoma, or thepredisposition to colon cancer by an assay that detects methylation inthe genes by restriction enzyme analysis, or using a nucleic acid basedmethod.

The disclosed invention provides a method and nucleic acids for theanalysis of colon carcinomas. It discloses a means of distinguishingbetween healthy and cancerous colon tissue. This provides a means forthe improved diagnosis, prognosis, staging and grading of colon cancer,at a molecular level, as opposed to currently used methods of arelatively subjective nature such as histological analysis. Furthermore,the disclosed invention presents improvements over the state of the artin that current methods of histological and cytological analysis requirethat the biopsy contain a sufficient amount of tissue. The methodaccording to the present invention can be used for classification ofminute samples.

The invention provides a method for detecting a colon cell proliferativedisorder characterised in that the target nucleic acid of one or moregenes taken from the group comprising estrogen receptor, p21, p27, p16,progesteron receptor, myoglobin, pcna, cdc2, c-erbB2, p53 and CEA arecontacted with a reagent or series of reagents capable of distinguishingbetween methylated and non methylated CpG dinucleotides within thetarget sequence.

The present invention makes available a method for ascertaining geneticand/or epigenetic parameters of genomic DNA. The method is for use inthe grading, staging, treatment and/or diagnosis of colon cancer. Themethod enables the analysis of cytosine methylations and singlenucleotide polymorphisms.

In one embodiment of the method the genomic DNA sample is first isolatedfrom tissue or cellular sources. Such sources may include cell lines,histological slides, body fluids, or tissue embedded in paraffin.Extraction may be by means that are standard to one skilled in the art,these include the use of detergent lysates, sonification and vortexingwith glass beads. Once the nucleic acids have been extracted the genomicdouble stranded DNA is used in the analysis.

In a preferred embodiment the DNA may be cleaved prior to the chemicaltreatment, this may be any means standard in the state of the art, inparticular with restriction endonucleases.

In the third step of the method, the genomic DNA sample is treated insuch a manner that cytosine bases which are unmethylated at the5′-position are converted to uracil, thymine, or another base which isdissimilar to cytosine in terms of hybridization behavior. This will beunderstood as ‘pretreatment’ hereinafter.

The above described treatment of genomic DNA is preferably carried outwith bisulfite (sulfite, disulfite) and subsequent alkaline hydrolysiswhich results in the conversion of non-methylated cytosine nucleobasesto uracil or to another base which is dissimilar to cytosine in terms ofbase pairing behavior.

In the fourth step of the method the bisulfite treated DNA is analysedusing one or a combination of several methods which are known in the artnamely real time PCR (Methyl Light assay), blocking oligonucleotides,methylation specific single nucleotide polymorphism extension(hereinafter referred to as MsSNuPE), methylation specific PCR(hereinafter referred to as MSP), and nucleic acid sequencing.

Fluorescence-based Real Time Quantitative PCR (Heid et al., Genome Res.6:986-994, 1996) employs a dual-labeled fluorescent oligonucleotideprobe (e.g. TaqMan™ PCR, using an ABI Prism 7700 Sequence DetectionSystem, Perkin Elmer Applied Biosystems, Foster City, Calif.) that ishybridized concurrently with oligonucleotide primers during acontinuosly monitered polymerase chain reaction. The TaqMan™ PCRreaction employs the use of a nonextendible interrogatingoligonucleotide, called a TaqMan™ probe, which is designed to hybridizeto a GpC-rich sequence located between the forward and reverseamplification primers. The TaqMan™ probe further comprises a fluorescent“reporter moiety” and a “quencher moiety” covalently bound to linkermoieties (e.g., phosphoramidites) attached to the nucleotides of theTaqMan™ oligonucleotide. For analysis of methylation within nucleicacids subsequent to bisulphite treatment it is required that the probebe methylation specific, as described in U.S. Pat. No. 6,331,393, alsoknown as the Methyl Light assay. Variations on the TaqMan™ detectionmethodology that are also suitable for use with the described inventioninclude the use of dual probe technology (Lightcycler™) or fluorescentamplification primers (Sunrise™ technology). Both these techniques maybe adapted in a manner suitable for use with bisulphite treated DNA, andmoreover for methylation analysis within CpG dinucleotides.

A further suitable method for the for the assessment of methylation byanalysis of bisulphite treated nucleic acids is the use of blockeroligonucleotides. The use of such oligonucleotides has been described inBioTechniques 23(4), 1997, 714-720 D. Yu, M. Mukai, Q. Liu, C. Steinman.Blocking probe oligonucleotides are hybridised to the bisulphite treatednucleic acid concurrently with the PCR primers. PCR amplification of thenucleic acid is terminated at the 5′ position of the blocking probe,thereby amplification of a nucleic acid is suppressed wherein thecomplementary sequence to the blocking probe is present. The probes maybe designed to hybridise to the bisulphite treated nucleic acid in amethylation status specific manner. For example, for detection ofmethylated nucleic acids within a population of unmethylated nucleicacids suppression of the amplification of nucleic acids which areunmethylated at the position in question would be carried out by the useof blocking probes comprising a ‘CG’ at the position in question, asopposed to a ‘CA’.

In a further preferred embodiment of the method the analysis is carriedout by the use of template directed oligonucleotide extension, such asMS SNuPE as described by Gonzalgo and Jones (Nucleic Acids Res.25:2529-2531).

In an alternative embodiment of the method the assessment of themethylation state fo the CpG dinucleotides may be carried out by PCRanalysis of the treated nucleic acid(s) using methylation specific PCR.Methylation specific primers (MSP) have been described, for example inU.S. Pat. No. 6,265,171 to Herman et al. MSP primers consist of anoligonucleotide specific for annealing to a nucleotide sequencecontaining at least one bisulphite treated CpG dinucleotide. Thereforethe sequence of said primers includes at least one CG, TG or CAdinucleotide. MSP primers specific for non methylated DNA contain a ‘T’at the 3′ position of the C position in the CpG. MSP primers generallycontain relatively few cytosines as these are converted by thebisulphite reaction. However when the primers are specifc for methylatedcytosine dinucleotides said cytosine positions are conserved within theprimer oligonucleotides.

The primers are extended by means of a polymerase and the resultantdouble stranded nucleic is denatured, preferably by means of heattreatment. Successive cycles of primer annealing, extension anddenaturation are carried out according to the polymerase chain reactionas described in U.S. Pat. No. 4,582,788 to Mullis.

In a further embodiment of the method the analysis is enabled bysequencing and subsequent sequence analysis of the amplificate generatedin the third step of the method (Sanger F., et al., 1977 PNAS USA 74:5463-5467).

In a particularly preferred embodiment, the method comprises thefollowing steps:

In the first step of the method the genomic DNA sample must be isolatedfrom tissue or cellular sources. Such sources may include cell lines,histological slides, body fluids, or tissue embedded in paraffin.Extraction may be by means that are standard to one skilled in the art,these include the use of detergent lysates, sonification and vortexingwith glass beads. Once the nucleic acids have been extracted the genomicdouble stranded DNA is used in the analysis.

In a preferred embodiment the DNA may be cleaved prior to the chemicaltreatment, this may be any means standard in the state of the art, inparticular with restriction endonucleases.

In the second step of the method, the genomic DNA sample is treated insuch a manner that cytosine bases which are unmethylated at the5′-position are converted to uracil, thymine, or another base which isdissimilar to cytosine in terms of hybridization behavior. This will beunderstood as ‘pretreatment’ hereinafter.

The above described treatment of genomic DNA is preferably carried outwith bisulfite (sulfite, disulfite) and subsequent alkaline hydrolysiswhich results in the conversion of non-methylated cytosine nucleobasesto uracil or to another base which is dissimilar to cytosine in terms ofbase pairing behavior.

In the third step fragments of the pretreated DNA are amplified, usingsets of primer oligonucleotides according to Seq ID 76 to 97, and a,preferably heat-stable polymerase. Because of statistical and practicalconsiderations, preferably more than ten different fragments having alength of 100-2000 base pairs are amplified. The amplification ofseveral DNA segments can be carried out simultaneously in one and thesame reaction vessel. Usually, the amplification is carried out by meansof a polymerase chain reaction (PCR).

The method may also be enabled by the use of alternative primers, thedesign of such primers is obvious to one skilled in the art. Theseshould include at least two oligonucleotides whose sequences are eachreverse complementary or identical to an at least 18 base-pair longsegment of the base sequences specified in the appendix (Seq. ID No.32through Seq. ID No.75). Said primer oligonucleotides are preferablycharacterized in that they do not contain any CpG dinucleotides. In aparticularly preferred embodiment of the method, the sequence of saidprimer oligonucleotides are designed so as to selectively anneal to andamplify, only the colon tissue specific DNA of interest, therebyminimizing the amplification of background or non relevant DNA. In thecontext of the present invention, background DNA is taken to meangenomic DNA which does not have a relevant tissue specific methylationpattern, in this case, the relevant tissue being colon tissue, bothhealthy and diseased.

According to the present invention, it is preferred that at least oneprimer oligonucleotide is bound to a solid phase during amplification.The different oligonucleotide and/or PNA-oligomer sequences can bearranged on a plane solid phase in the form of a rectangular orhexagonal lattice, the solid phase surface preferably being composed ofsilicon, glass, polystyrene, aluminum, steel, iron, copper, nickel,silver, or gold, it being possible for other materials such asnitrocellulose or plastics to be used as well.

The fragments obtained by means of the amplification can carry adirectly or indirectly detectable label. Preferred are labels in theform of fluorescence labels, radionuclides, or detachable moleculefragments having a typical mass which can be detected in a massspectrometer, it being preferred that the fragments that are producedhave a single positive or negative net charge for better detectabilityin the mass spectrometer. The detection may be carried out andvisualized by means of matrix assisted laser desorption/ionization massspectrometry (MALDI) or using electron spray mass spectrometry (ESI).

The amplificates obtained in the third step of the method aresubsequently hybridized to an array or a set of oligonucleotides and/orPNA probes. In this context, the hybridization takes place in the mannerdescribed in the following. The set of probes used during thehybridization is preferably composed of at least 10 oligonucleotides orPNA-oligomers. In the process, the amplificates serve as probes whichhybridize to oligonucleotides previously bonded to a solid phase. Inaparticularly preferred embodiment, the oligonucleotides are taken fromthe group comprising Seq IDs 98 to 523. The non-hybridized fragments aresubsequently removed. Said oligonucleotides contain at least one basesequence having a length of 10 nucleotides which is reversecomplementary or identical to a segment of the base sequences specifiedin the appendix, the segment containing at least one CpG dinucleotide.The cytosine of the CpG dinucleotide is the 5^(th) to 9^(th) nucleotidefrom the 5′-end of the 10-mer. One oligonucleotide exists for each CpGdinucleotide.

In the next step of the method, the non-hybridized amplificates areremoved.

In the final step of the method, the hybridized amplificates aredetected. In this context, it is preferred that labels attached to theamplificates are identifiable at each position of the solid phase atwhich an oligonucleotide sequence is located.

According to the present invention, it is preferred that the labels ofthe amplificates are fluorescence labels, radionuclides, or detachablemolecule fragments having a typical mass which can be detected in a massspectrometer. The mass spectrometer is preferred for the detection ofthe amplificates, fragments of the amplificates or of probes which arecomplementary to the amplificates, it being possible for the detectionto be carried out and visualized by means of matrix assisted laserdesorption/ionization mass spectrometry (MALDI) or using electron spraymass spectrometry (ESI). The produced fragments may have a singlepositive or negative net charge for better detectability in the massspectrometer.

The aforementioned method is preferably used for ascertaining geneticand/or epigenetic parameters of genomic DNA.

In order to enable this method, the invention further provides thechemically modified DNA of the genes estrogen receptor, p21, p27, p16,progesteron receptor, myoglobin, pcna, cdc2, c-erbB2, p53 and CEA aswell as oligonucleotides and/or PNA-oligomers for detecting cytosinemethylations. The present invention is based on the discovery thatgenetic and epigenetic parameters and, in particular, the cytosinemethylation patterns of genomic DNA are particularly suitable forcharacterisation, grading, staging, and/or diagnosis of colon cancer.

The nucleic acids according to the present invention of Seq. ID No.12through Seq. ID No. 523 can be used for characterisation, grading,staging and/or diagnosis of genetic and/or epigenetic parameters ofgenomic DNA.

This objective is achieved according to the present invention using anucleic acid containing a sequence of at least 18 bases in length of thechemically pretreated genomic DNA according to one of Seq. ID No.32through Seq. ID No.75 and sequences complementary thereto.

The chemically modified nucleic acid could heretofore not be connectedwith the ascertainment of disease relevant genetic and epigeneticparameters.

The object of the present invention is further achieved by anoligonucleotide or oligomer for the analysis of pretreated DNA, fordetecting the genomic cytosine methylation state, said oligonucleotidecontaining at least one base sequence having a length of at least 10nucleotides which hybridizes to a pretreated genomic DNA according toSeq. ID No.32 through Seq. ID No. 75. The oligomer probes according tothe present invention constitute important and effective tools which,for the first time, make it possible to ascertain specific genetic andepigenetic parameters of colon cancers, in particular, for use incharacterisation, grading, staging, and/or diagnosis of colon cancer.The base sequence of the oligomers preferably contains at least one CpGdinucleotide. The probes may also exist in the form of a PNA (peptidenucleic acid) which has particularly preferred pairing properties.Particularly preferred are oligonucleotides according to the presentinvention in which the cytosine of the CpG dinucleotide is the5^(th)-9^(th) nucleotide from the 5′-end of the 13-mer; in the case ofPNA-oligomers, it is preferred for the cytosine of the CpG dinucleotideto be the 4^(th)-6^(th) nucleotide from the 5′-end of the 9-mer.

The oligomers according to the present invention are normally used in socalled “sets” which contain at least one oligomer for each of the CpGdinucleotides of the sequences of Seq. ID No. 32 to Seq. ID No. 75.Preferred is a set which contains at least one oligomer for each of theCpG dinucleotides from one of Seq. ID No. 32 to Seq. ID No. 75.

In the case of the sets of oligonucleotides according to the presentinvention, it is preferred that at least one oligonucleotide is bound toa solid phase. It is further preferred that all the oligonucleotides ofone set are bound to a solid phase.

The present invention moreover relates to a set of at least 10 n(oligonucleotides and/or PNA-oligomers) used for detecting the cytosinemethylation state in chemically pretreated genomic DNA (Seq. ID No.32 toSeq. ID No.75 No.75 and sequences complementary thereto). These probesenable characterisation, grading, staging and/or diagnosis of geneticand epigenetic parameters of colon cancer. Furthermore, the probesenable the diagnosis of predisposition to colon cancer. The set ofoligomers may also be used for detecting single nucleotide polymorphisms(SNPs) in pretreated genomic DNA according to one of Seq. ID No. 32 toSeq. ID No. 75.

According to the present invention, it is preferred that an arrangementof different oligonucleotides and/or PNA-oligomers (a so-called “array”)made available by the present invention is present in a manner that itis likewise bound to a solid phase. This array of differentoligonucleotide- and/or PNA-oligomer sequences can be characterized inthat it is arranged on the solid phase in the form of a rectangular orhexagonal lattice. The solid phase surface is preferably composed ofsilicon, glass, polystyrene, aluminum, steel, iron, copper, nickel,silver, or gold. However, nitrocellulose as well as plastics such asnylon which can exist in the form of pellets or also as resin matricesare possible as well.

Therefore, a further subject matter of the present invention is a methodfor manufacturing an array fixed to a carrier material for the grading,staging, and/or diagnosis of colon cancer, in which method at least oneoligomer according to the present invention is coupled to a solid phase.Methods for manufacturing such arrays are known, for example, from U.S.Pat. No. 5,744,305 by means of solid-phase chemistry and photolabileprotecting groups.

A further subject matter of the present invention relates to a DNA chipfor the characterisation, grading, staging, and/or diagnosis of coloncancer. Furthermore the DNA chip enables the diagnosis of predispositionto colon cancer. The DNA chip contains at least one nucleic acidaccording to the present invention. DNA chips are known, for example, inU.S. Pat. No. 5,837,832.

Moreover, a subject matter of the present invention is a kit which maybe composed, for example, of a bisulfite-containing reagent, a set ofprimer oligonucleotides containing at least two oligonucleotides whosesequences in each case correspond or are complementary to a 18 base longsegment of the base sequences specified in the appendix (Seq. ID No.32through Seq. ID No.75), oligonucleotides and/or PNA-oligomers as well asinstructions for carrying out and evaluating the described method.However, a kit along the lines of the present invention can also containonly part of the aforementioned components.

The oligomers according to the present invention or arrays thereof aswell as a kit according to the present invention are intended to be usedfor the characterisation, grading, staging and/or diagnosis of coloncancer, or diagnosis of predisposition to colon cancer. According to thepresent invention, the method is preferably used for the analysis ofimportant genetic and/or epigenetic parameters within genomic DNA, inparticular for use in characterisation, grading, staging and/ordiagnosis of colon cancer, and predisposition to colon cancer.

The methods according to the present invention are used, for example,for characterisation, grading, staging and/or diagnosis of colon cancer.

A further embodiment of the invention is a method for the analysis ofthe methylation status of genomic DNA without the need for chemicalpretreatment. In the first step of the method the genomic DNA samplemust be isolated from tissue or cellular sources. Such sources mayinclude cell lines, histological slides, body fluids, or tissue embeddedin paraffin; for example, brain, central nervous system or lymphatictissue. Extraction may be by means that are standard to one skilled inthe art, these include the use of detergent lysates, sonification andvortexing with glass beads. Once the nucleic acids have been extractedthe genomic double stranded DNA is used in the analysis.

In a preferred embodiment the DNA may be cleaved prior to the chemicaltreatment, this may be any means standard in the state of the art, inparticular with restriction endonucleases. In the second step, the DNAis then digested with methylation sensitive restriction enzymes. Thedigestion is carried out such that hydrolysis of the DNA at therestriction site is informative of the methylation status of a specificCpG dinucleotide.

In the third step the restriction fragments are amplified. In apreferred embodiment this is carried out using a polymerase chainreaction.

In the final step the amplificates are detected. The detection may be byany means standard in the art, for example, but not limited to, gelelectrophoresis analysis, hybridisation analysis, incorporation ofdetectable tags within the PCR products, DNA array analysis, MALDI orESI analysis.

The present invention moreover relates to the diagnosis and/or prognosisof events which are disadvantageous or relevant to patients orindividuals in which important genetic and/or epigenetic parameterswithin genomic DNA, said parameters obtained by means of the presentinvention may be compared to another set of genetic and/or epigeneticparameters, the differences serving as the basis for a diagnosis and/orprognosis of events which are disadvantageous or relevant to patients orindividuals.

In the context of the present invention the term “hybridization” is tobe understood as a bond of an oligonucleotide to a completelycomplementary sequence along the lines of the Watson-Crick base pairingsin the sample DNA, forming a duplex structure.

The term “functional variants” denotes all DNA sequences which arecomplementary to a DNA sequence, and which hybridize to the referencesequence under stringent conditions.

In the context of the present invention, “genetic parameters” aremutations and polymorphisms of genomic DNA and sequences furtherrequired for their regulation. To be designated as mutations are, inparticular, insertions, deletions, point mutations, inversions andpolymorphisms and, particularly preferred, SNPs (single nucleotidepolymorphisms).

In the context of the present invention, “epigenetic parameters” are, inparticular, cytosine methylations and further chemical modifications ofDNA bases of genomic DNA and sequences further required for theirregulation. Further epigenetic parameters include, for example, theacetylation of histones which, cannot be directly analyzed using thedescribed method but which, in turn, correlates with the DNAmethylation.

In the following, the present invention will be explained in greaterdetail on the basis of the sequences and examples without being limitedthereto.

Seq. ID 1 to 11 represent the genomic DNA of genes estrogen receptor,p21, p27, p16, progesteron receptor, myoglobin, pcna, cdc2, c-erbB2, p53and CEA. These sequences are derived from Genbank and will be taken toinclude all minor variations of the sequence material which arecurrently unforseen, for example, but not limited to, minor deletionsand SNPs.

Sequence ID 12 to 31 represent segments of genomic DNA which areparticularly useful for the determination of colon cell proliferativedisorder.

Sequence ID 32 to 75 exhibit the chemically pretreated sequence of genesestrogen receptor, p21, p27, p16, progesteron receptor, myoglobin, pcna,cdc2, c-erbB2, p53 and CEA. These sequences will be taken to include allminor variations of the sequence material which are currently unforseen,for example, but not limited to, minor deletions and SNPs.

Sequences having even sequence numbers (e.g., Seq. ID No. 32, 34, 36, .. . ) exhibit in each case sequences of chemically pretreated genomicDNAs.

Sequences having odd sequence numbers (e.g., Seq. ID No. 33, 35, 37 . .. ) exhibit in each case the sequences of chemically pretreated genomicDNAs. Said genomic DNAs are complementary to the genomic DNAs from whichthe preceeding sequence was derived (e.g., the complementary sequence tothe genomic DNA from which Seq. ID No.32 is derived is the genomicsequence from which Seq. ID No.33 is derived, the complementary sequenceto the genomic DNA from which Seq. ID No.33 is derived is the sequencefrom which Seq. ID No.34 is derived, etc.)

Sequence ID 76 to 97 exhibit the sequence of primer oligonucleotides forthe amplification of chemically pretreated DNA according to Sequence IDs32 to 75.

Sequence IDs 98 to 523 exhibit the sequence of oligomers which areparticularly useful for the analysis of CpG positions within chemicallypretreated DNA according to Sequence IDs 32 to 75.

The following examples describe the invention in detail without limitingthe scope of the invention.

EXAMPLE 1 Description of PCR

The single gene PCR reaction was performed using a thermocycler(Epperdorf GmbH) using 10 ng of bisulfite treated DNA, 6 pmole of eachprimer, 200 μM of each dNTP, 1.5 mM MgCl₂ and 1 U of HotstartTaq (QiagenAG). The other conditions were as recommended by the Taq polymerasemanufacturer. Single genes were amplified by PCR performing a firstdenaturation step for 14 min at 96° C., followed by 39 cycles (60 sec at96° C., 45 sec at 55° C. 75 sec at 72° C.) and a subsequent finalelongation of 10 min at 72° C. The bisulfite DNA was prepared accordingto a published procedure from genomic DNA individually isolated from 12matched samples of adenocarzinoma of the colon and healthy colon tissue.The genomic DNA was isolated using the wizzard DNA isolation kit(Promega, Madison).

EXAMPLE 2 Methylation Analysis of Gene p16

The following example relates to a fragment of the gene p16 in which aspecific CG dinucleotide is to be analyzed for methylation.

In the first step, a genomic sequence is treated using bisulfite(hydrogen sulfite, disulfite) in such a manner that all cytosines whichare not methylated at the 5-position of the base are modified in such amanner that a different base is substituted with regard to the basepairing behavior while the cytosines methylated at the 5-position remainunchanged.

If bisulfite solution is used for the reaction, then an addition takesplace at the non-methylated cytosine bases. Moreover, a denaturatingreagent or solvent as well as a radical interceptor must be present. Asubsequent alkaline hydrolysis then gives rise to the conversion ofnon-methylated cytosine nucleobases to uracil. The chemically convertedDNA is then used for the detection of methylated cytosines. In thesecond method step, the treated DNA sample is diluted with water or anaqueous solution. Preferably, the DNA is subsequently desulfonated. Inthe third step of the method, the DNA sample is amplified in apolymerase chain reaction, preferably using a heat-resistant DNApolymerase. In the present case, cytosines of the gene p16 are analyzed.To this end, a defined fragment having a length of 598 bp is amplifiedwith the specific primer oligonucleotides TTGAAAATTAAGGGTTGAGG (SequenceID 82) and CACCCTCTAATAACCAACCA. (Sequence ID No. 83)The amplificate serves as a sample which hybridizes to anoligonucleotide previously bound to a solid phase, forming a duplexstructure, for example TAAGTGTTCGGAGTTAAT (SEQ ID NO: 238), the cytosineto be detected being located at position 439 of the amplificate. Thedetection of the hybridization product is based on Cy3 and Cy5fluorescently labelled primer oligonucleotides which have been used forthe amplification. A hybridization reaction of the amplified DNA withthe oligonucleotide takes place only if a methylated cytosine waspresent at this location in the bisulfite-treated DNA as shown forhealthy tissue in FIG. 1A. Thus, the methylation status of the specificcytosine to be analyzed is inferred from the hybridization product.

In order to verify the methylation status of the position, a sample ofthe amplificate is further hybridized to another oligonucleotidepreviously bonded to a solid phase. Said olignonucleotide is identicalto the oligonucleotide previously used to analyze the methylation statusof the sample, with the exception of the position in question. At theposition to be analysed said oligonucleotide comprises a thymine base asopposed to a cytosine base i.e TAAGTGTTTGGAGTTAAT (SEQ ID NO: 239).Therefore, the hybridisation reaction only takes place if anunmethylated cytosine was present at the position to be analysed asshown for tumor tissue in FIG. 1B.

EXAMPLE 3 Differentiation Between Colon Tumour and Healthy Colon Tissue

Differentiation of healthy samples and adenocarzinoma tumours. Fortumour class prediction between healthy and tumor tissue we used aSupport Vector Machine (SVM) on a set of selected CpG sites (F. Model,P. Adorjan, A. Olek, C. Piepenbrock, Feature selection for DNAmethylation based cancer classification. Bioinformatics. 2001 June;17Suppl 1:S157-64.). First we ranked the CpG sites for a given separationtask by their significance of the difference between the two classmeans. The significance of each CpG was estimated by a two sample t-test(W, Mendenhall, T, Sincich, Statistics for engineering and the sciences(Prentice-Hall, New Jersey 1995).

In order to relate the methylation patterns to a adenocarcinoma tumour,it is initially required to comparatively analyze the DNA methylationpatterns of healthy tissue and adenocarzinoma tumours tissue (FIGS. 2Aand B). These analyses were carried out, analogously to Examples 1. Theresults obtained in this manner are stored in a database and the CpGdinucleotides which are methylated differently between the two groupsare identified. This can be carried out by determining individual CpGmethylation rates as can be done, for example, by sequencing, which is arelatively imprecise method of quantifying methylation at a specificCpG, or else, in a very precise manner, by a methylation-sensitive“primer extension reaction”. In a particularly preferred variant, asillustrated in the preceeding examples the methylation status ofhundreds or thousands of CpGs may be analysed on an oligomer array. Itis also possible for the patterns to be compared, for example, byclustering analyses which can be carried out, for example, by acomputer.

A panel of genomic fragments of 11 different genes (listed in Table 1)were bisulphite treated and amplified by singleplex PCRs according toExample 1. However, as will be obvious to one skilled in the art, it isalso possible to use other primers that amplify the genomic, bisulphitetreated DNA in an adequate manner, and/or to carry out the PCRs in amultiplex format. However the primer oligonucleotide pairs as listed inTable 1 are particularly preferred. In order to differentiateadenocarzinoma tumour from healthy control samples optimal results wereobtained by including at least 6 CpG dinucleotides, the most informativeCpG positions for this discrimination being located within the p16, p53,CEA, c-erbB2 and estrogen receptor genes (cf. FIG. 2, Tab1). Inaddition, the majority of the analysed CpG dinucleotides of the panelshowed different methylation patterns between the two phenotypes. Theresults prove that methylation fingerprints are capable of providingdifferential diagnosis of adenocarzinoma tumours and could therefore beapplied in a large number clinical situations

For class prediction a SVM was trained on the most significant CpGpositions, where the optimal number of CpG sites depends on thecomplexity of the separation task. Implementation of the SVM used theSequential Minimal Optimization algorithm to find the 1-norm soft marginseparating hyperplane (N. Christianini, J. Shawe-Taylor, An Introductionto Support Vector Machines, Cambridge University Press, Cambridge 2000).The box constraint was set to C=10. Generalization performance wasestimated by averaging over 50 cross validation runs on randomlypermutated samples partitioned into 8 groups.

EXAMPLE 4 Analysis of the Methylation Status of the most Informative CpGPositions of the Genes c-erbB2, p53, CEA, p16 and ER1

The methylation status of the most informative CpG positions of the genefragments of genes c-erbB2, p53, CEA, p16 and ER1 are shown in thisexample. Corresponding to Example 2, where the methylation status isdemonstrated by spots, Table 2 describes in a more detailed way themethylation status of different gene fragments of various patients bycalculating the methylation status of colon tumour and healthy colontissue. The first column indicates the specific gene fragment, thesecond column describes the investigated CpG Oligonukleotide, the thirdcolumn depicts the diagnosis of the investigated tissue (T=tumor,H=healthy) and columns 4 to 17 show the logarithm of the ratio ofv thefluorescence signal of the CG oligonucleotide versus TG oligonucleotideof colon tumour and healthy colon tissue of 14 different patients. Forexample, a comparison of the methylation status of gene p16, patient 11,shows that the healthy tissue is less methylated compared to the tumourtissue for this sample. The opposite ratio can be observed, for example,for gene c-erbB2 for patient 11. In this case the tumour sample is moremethylated than the healthy, sample. The analyzed CpG positions showthat the genes p53, CEA, p16 and ER1 are hypermethylated, whereasc-erbB2 is hypomethylated in most of the tumour samples compared withthe healthy controls.

EXAMPLE 5 Identification of the Methylation Status of CpG Sites of GenesCEA and p16 by Methylation Sensitive Restriction Enzyme Digest

In the CEA gene, a defined fragment having a length of 351 bp, whichcontains 7 CpG sites, is amplified with the specific primeroligonucleotides TGGTTAAATGTGTGGGAGAT (Sequence ID 524) andTCCTGAGTGATGTCTGTGTG (Sequence ID No. 525) and in the p16 gene, adefined fragment having a length of 391 bp, which contains 26 CpG sites,is amplified with the specific primer oligonucleotidesATGACACCAAACACCCCGAT (Sequence ID 526) and CTGTCCCTCAAATCCTCTG (SequenceID No. 527). CGCG for gene CEA with Cytosins at positions 127 and 129 ofthe amplificate and CGCG for gene p16 with Cytosins at positions 362 and364 of the amplificate, are located in a SacII restriction enzymerecognition sequence, CCGCGG. The cleavage of SacII is blocked bymethylation of at least one of the two CpG dinucleotides.

The genomic DNA isolated from adenocarzinoma of colon tissue and fromhealthy colon tissue was hydrolysed by SacII as recommended by themanufacturer (New England Biolabs GmbH).

10 ng of the SacII restricted DNA was used as template for theamplification of the above indicated CEA and p16 gene fragments. The PCRreaction was performed using a thermocycler (Eppendorf GmbH) using 10 ngof DNA, 6 pmole of each primer, 200 μM of each dNTP, 1.5 mM MgCl2 and 1U of HotstartTaq (Qiagen AG). The other conditions were as recommendedby the Taq polymerase manufacturer. Using the above mentioned primers,gene fragments were amplified by PCR performing a first denaturationstep for 14 min at 96° C., followed by 30-45 cycles (step 2:60 sec at96° C., step 3:45 sec at 55° C., step 4: 75 sec at 72° C.) and asubsequent final elongation of 10 min at 72° C. The presence of PCRproducts was analysed by agrarose gel electrophoresis.

PCR products were detectable with SacII hydrolyzed DNA isolated fromcolon cancer tissue, when step 2 to step 4 of the cycle program wererepeated 34, 37, 39, 42 and 45 fold. In contrast PCR products were onlydetectable with SacII hydrolyzed DNA isolated from healthy colon tissuewhen step 2 to step 4 of the cycle program were repeated 42 and 45 fold.These results indicate that at least one of CpG positions located withinthe SacII recognition sequence of the analysed CEA and the p16 genefragment showed a higher methylation status in cancer samples comparedto the healthy control.

DESCRIPTION OF FIGURES

FIG. 1

FIG. 1 shows the hybridisation of fluorescent labelled amplificates to asurface bound olignonucleotide. Sample A being from healthy tissue andsample B being from colon adenocarzinoma tissue. Fluorescence at a spot,denoted by an arrow, indicates hybridisation of the amplificate againstthe olignonucleotide. Hybridisation to a CG olignonucleotide with thesequence TAAGTGTTCGGAGTTAAT (SEQ ID NO: 238) denotes methylation at thecytosine position being analysed, hybridisation to a TG olignonucleotidewith the sequence TAAGTGTTTGGAGTTAAT (SEQ ID NO: 239) denotes nomethylation at the cytosine position being analysed. It can be seen thatsample A was umethylated for CG positions of the amplificate of gene p16whereas in comparison sample B had a higher degree of methylation at thesame position.

FIG. 2

Differentiation of colon tumour (A) from healthy colon tissue (B). Highprobability of methylation corresponds to red, uncertainty to black andlow probability to green. The labels on the left side of the plot aregene (e.g. for the topmost: 2064) and CpG (e.g. for the topmost: 1485A)identifiers. The hybridisation was carreid out with Cy5 labelledamplificates generated by singlplex PCR reactions using primeroligonucleotides as shown in Table 1. The labels on the right side givethe significance (p-value, T-test) of the difference between the meansof the two groups. Each row corresponds to a single CpG and each columnto the methylation levels of one sample. CpGs are ordered according totheir contribution to the distinction to the differential diagnosis ofthe two lesions with increasing contribution from top to bottom.

TABLE 1 List of genes, reference numbers (ID) according FIG. 2 andprimer oligonucleotides according to Example 2 and FIGS. 1 and 2. GeneGene Seq-ID Accession no Primer Seq-ID PCR primer Primer Seq-ID PCRprimer Estrogen- 1 NM_000125 76 AGGAGGGGGAATTAAATAGA 77ACAATAAAACCATCCCAAATAC receptor P21 2 NM_000389 78 GGATTAGTGGGAATAGAGGTG79 AAACCCAAACTCCTAACTACC P27 3 NM_004064 80 GTGGGGAGGTAGTTGAAGA 81ATACACCCCTAACCCAAAAT P16 4 NM_000077 82 TTGAAAATTAAGGGTTGAGG 83CACCCTCTAATAACCAACCA Progesteron- 5 NM_000926 84 GAGGGGGTAGTGGAATTTAG 85CCTTTACCTTCAACTCAATCA receptor Myoglobin 6 NM_005368 86GTTTTTGGTAAAGGGGTAGAA 87 CCTAAAATATCAACCTCCACCT PCNA 7 NM_002592 88TTTTTAGGTTGTAAGGAGGTTTT 89 TAAATACCTCCAACACCTTTCT CDC2 8 NM_001786 90ATTAGAAGTGAAAGTAATGGAATTT 91 TCAATTTCCAAAAACCAAC c-erbB2 9 NM_004448 92GGAGGGGGTAGAGTTATTAGTT 93 TATACTTCCTCAAACAACCCTC P53 10 NM_000546 94GATTGGGTAAGTTTTTGATTGA 95 AAATCTCCCAACAATACAACTC CEA 11 NM_004363 96GTTTAGGATGGGATTAAGTGTG 97 AATCAAATATCCCCAAATACAA

TABLE 2 log (fluorescence OGoligo/fluorescence TGoligo) of matched pairof colon tumor (T) and healthy colon tissue (H)^(a) gene OpG Diagnosis11 3 9 1 8 2 4 13 12 14 15 5 10 6 c-erbB2 2064:148 T −1.07 −1.72 −1.11−1.53 −1 −1.3 −1.63 −1.01 −1.64 −1.22 −1.33 −0.88 −1.57 −1.22 H −0.82−1.09 −1.34 −1.17 −0.75 −1.09 −1.36 −0.89 −0.88 −0.72 −1.06 −0.93 −1.43−0.7 p53 2317:122 T −2.17 −0.58 −2.37 −1.91 −2.08 −0.32 −0.3 −1.63 −2.19−1.87 −1.71 −4.31 −3.15 −1 H −4.03 −3.01 −3.14 −3.83 −1.53 −2.29 −1.86−2.96 −2.76 −4.09 −3.44 −4.77 −2.33 −3.97 p53 2317:153 T −2.38 −2.84−2.77 −2.57 −2.93 −2.44 −2.89 −3.12 −2.77 −2.32 −2.7 −2.89 −2.13 −2.29 H−3.36 −3.36 −3.17 −3.32 −3.15 −3.47 −2 −2.94 −3.8 −3.77 −3.67 −4.12 −2.2−3.12 CEA 2398:176 T −2.76 −1.84 −3.7 −2.42 −2.59 −0.83 −2.14 −1.96−2.86 −4.02 −2.76 −5.71 −5.07 −2.33 H −4.32 −2.64 −4.72 −3.9 −4.43 −4.85−2.95 −3 −2.67 −4.19 −2.92 −4.63 −4.53 −3.64 CEA 2398:227 T −3.35 −2.15−3.83 −3.88 −4.02 −2.95 −2.81 −3.98 −3.9 −4.01 −4.75 −4.64 −4.34 −3.15 H−4.7 −4.37 −5.1 −5.77 −4.7 −4.64 −3.33 −3.65 −5.48 −5.2 −5.32 −5.75−3.84 −5.4 p16 2035:181 T −1.64 −1.99 −2.66 −2.6 −3.78 −1.07 −2.21 −2.18−3.24 −1.8 −2.19 −3.21 −3.25 −2.13 H −2.74 −3.02 −4.09 −3.52 −3.74 −3.87−2.88 −2.76 −3.28 −2.27 −3.19 −3.38 −3.88 −3.49 ER1 41:2912 T −0.4 −0.37−1.23 −0.96 −1.36 −0.47 0.37 −0.34 −0.76 −0.85 −0.56 −1.32 −1.53 −1.33 H−0.8 −1.25 −2.1 −1.23 −1.52 −1.38 −0.44 −0.93 −1.26 −1.55 −1.21 −1.55 −1−1.45 ER1 41:2860 T −1.06 −0.77 −2.06 −1.8 −1.7 −0.53 −1.52 −1.82 −1.19−1.59 −0.82 −1.6 −1.85 −1.69 H −1.83 −2.03 −2.05 −2.22 −2.36 −2.13 −2.26−1.72 −0.91 −2.11 −1.6 −2.05 −1.9 −2.14 ER1 41:2428 T 0.02 0.84 −1.41−1.22 −1.39 0.67 −0.13 −0.33 −0.66 −1.41 0.22 −1.78 −1.61 −1 H −0.97−0.86 −1.61 −1.36 −1.02 −1.78 −0.88 −1.05 −1.65 −1.29 −1.19 −1.53 −1.45−1.96 ER1 41:2849 T −0.86 −0.43 −1.55 −0.98 −1.22 −0.97 −2.16 −1.2 −0.66−1.07 −0.54 −1.59 −1.45 −0.78 H −1.11 −1.04 −1.97 −1.27 −2.06 −2.21−2.77 −1.01 −1.08 −1.59 −1.08 −1.9 −2.02 −1.76^(a)the values indicate the mean of at least 12 OG/TG oligo pairsanalysed in 3 independent chip hybridisation

1. A method to determine the methylation status of CpG dinucleotideswithin one or more of the genes estrogen receptor, p21, p27, p 16,progesterone receptor, myoglobin, pcna, cdc2, c-erB2, p53 and CEAcomprising contacting the target nucleic acid in a biological samplewith at least one reagent or series of reagents wherein said reagent orseries of reagents distinguishes between methylated and non methylatedCpG dinucleotides within the target nucleic acid and concluding from themethylation status of one or more of said CpG positions on the presenceor absence of a colon cell proliferative disorder.
 2. A method accordingto claim 1 comprising the following steps: obtaining a biological samplecontaining genomic DNA extracting the genomic DNA in the genomic DNAsample, cytosine bases which are unmethylated at the 5-position areconverted, by treatment, to uracil or another base which is dissimilarto cytosine in terms of base pairing behavior; fragments of thepretreated genomic DNA are amplified using sets of primeroligonucleotides according to Seq ID 76 to Seq ID 97 and a polymerase,the amplificates carrying a detectable label; detection of the fragmentsIdentification of the methylation status of one or more cytosinepositions
 3. A method according to claim 2, characterized in that thereagent is a solution of bisulfite, hydrogen sulfite or disulfite.
 4. Amethod as recited in one of claims 2 and 3, characterized in that theamplification is carried out by means of the polymerase chain reaction(PCR).
 5. A method as recited in one of the claims 2 to 3, characterizedin that more than ten different fragments having a length of 100-2000base pairs are amplified.
 6. A method as recited in one of the claims 2to 3, characterized in that the amplification of several DNA segments iscarried out in one reaction vessel.
 7. A method as recited in one of theclaims 2 to 3, characterized in that the polymerase is a heat-resistantDNA polymerase.
 8. A method as recited in one of the claims 2 to 3,characterized in that the labels of the amplificates are fluorescencelabels.
 9. A method as recited in one of claims 2 to 3, characterized inthat the labels of the amplificates are radionuclides.
 10. A methodaccording to one of claims 2 to 3, characterized in that eachamplificate is detected by hybridization to an oligonucleotide orpeptide nucleic acid (PNA)-oligomer.
 11. A method according to claim 10,characterized in that the olignonucleotide or peptide nucleic acid(PNA)-oligomer is taken from the group comprising Seq ID 98 to
 523. 12.A method as recited in one of claims 2 to 3, characterized in that thelabels of the amplificates are detachable molecule fragments having atypical mass which are detected in a mass spectrometer.
 13. A method asrecited in one of claims 2 to 3, characterized in that the amplificatesor fragments of the amplificates are detected in the mass spectrometer.14. A method as recited in claim 12, characterized in that the producedfragments have a single positive or negative net charge for betterdetectability in the mass spectrometer.
 15. A method as recited in claim12, characterized in that detection is carried out and visualized bymeans of matrix assisted laser desorption/ionization mass spectrometry(MALDI) or using electron spray mass spectrometry (ESI).
 16. A method asrecited in claim 2, characterized in that the amplification steppreferentially amplifies DNA which is of particular interest in healthyand/or diseased colon tissues, based on the specific genomic methylationstatus of colon tissue, as opposed to background DNA.
 17. A methodaccording to claim 1 comprising the following steps; a) obtaining abiological sample containing genomic DNA b) extracting the genomic DNA,c) digesting the target nucleic acids with one or more methylationsensitive restriction enzymes, d) amplification of the DNA digest and e)detection of the amplificates.
 18. A method according to claim 17wherein the target nucleic acids comprise one or more sequences takenfrom the group according to Seq ID 12 to Seq ID 31 or sequenceshybridising thereto and fragments thereof.
 19. A method as recited inone of claims 17 or 18, characterized in that the amplification iscarried out by means of the polymerase chain reaction (PCR).
 20. Amethod as recited in one of claims 17 to 18, characterized in that theamplification of several DNA segments is carried out in one reactionvessel.
 21. A method as recited in one of claims 17 to 18, characterizedin that the polymerase is a heat-resistant DNA polymerase.
 22. Anisolated nucleic acid of the pretreated genomic DNA according to one ofthe sequences taken from the group comprising Seq. ID No.32 to Seq. IDNo.75 and sequences complementary thereto.
 23. An oligomer, inparticular an oligonucleotide or peptide nucleic acid (PNA)-oligomer,said oligomer comprising at least one base sequence of at least 10nucleotides which hybridizes to or is identical to a pretreated genomicDNA according to one of the Seq. ID No. 32 to Seq. ID No 75 according toclaim
 22. 24. An oligomer or peptide nucleic acid (PNA)-oligomer asrecited in claim 23, wherein the base sequence includes at least one CpGdinucleotide sequence.
 25. An oligomer or peptide nucleic acid(PNA)-oligomer as recited in claim 23, characterized in that thecytosine of the at least one CpG dinucleotide is/are locatedapproximately in the middle third of the oligomer.
 26. An oligomer orpeptide nucleic acid (PNA)-oligomer, in particular an oligonucleotide,according to one of the sequences taken from the group comprising Seq.ID No.98 to Seq. ID No.
 523. 27. A set of oligomers or peptide nucleicacid (PNA)-oligomers, comprising at least two oligomers according to anyof claims 22 to
 26. 28. A set of oligomers or peptide nucleic acid(PNA)-oligomers as recited in claim 27, comprising oligomers fordetecting the corresponding genomic methylation state of all CpGdinucleotides within one of the sequences according to Seq. ID Nos. 32to 75, and sequences complementary thereto.
 29. A set of at least twooligonucleotides or peptide nucleic acid (PNA)-oligomers as recited inclaim 23, as primer oligonucleotides for the amplification of DNAsequences of one of Seq. ID 32 to Seq. ID 75 and/or sequencescomplementary thereto and segments thereof.
 30. A set ofoligonucleotides or peptide nucleic acid (PNA)-oligomers as recited inone of claims 22 and 23, characterized in that at least oneoligonucleotide is bound to a solid phase.
 31. Use of a set of oligomersor peptide nucleic acid (PNA)-oligomers according to one of thesequences taken from the group comprising Seq. ID No. 32 to Seq. ID No.75 and sequences complementary thereto as probes for determining thecytosine methylation state and/or single nucleotide polymorphisms (SNPs)of a corresponding genomic DNA by analysis of a chemically pretreatedgenomic DNA according to claim
 2. 32. Use of a pretreated genomic DNAaccording to claim 22 for the determination of the methylation status ofa corresponding genomic DNA and/or detection of single nucleotidepolymorphisms (SNPs).
 33. A method for manufacturing an arrangement ofdifferent oligomers or peptide nucleic acid (PNA)-oligomers (array) foranalyzing diseases associated with the corresponding genomic methylationstatus of the CpG dinucleotides within one of the Seq. ID 32 to Seq. ID75 and sequences complementary thereto, wherein at least one oligomeraccording to any of the claims 22 to 26 is coupled to a solid phase. 34.An arrangement of different oligomers or peptide nucleic acid(PNA)-oligomers (array) obtainable according to claim
 33. 35. An arrayof different oligonucleotide- and/or PNA-oligomer sequences as recitedin claim 34, characterized in that these are arranged on a plane solidphase in the form of a rectangular or hexagonal lattice.
 36. A DNA/PNAarray for the analysis of prostate cell proliferative disordersassociated with the methylation state of genes comprising at least onenucleic acid according to claim
 22. 37. An array as recited in any ofthe claims 34 to 36, characterized in that the solid phase surface iscomposed of silicon, glass, polystyrene, aluminium, steel, iron, copper,nickel, silver, or gold.
 38. Use of a method according to claim 1 forthe characterisation, classification, diagnosis and differentiation ofcolon cell proliferative disorders.
 39. A kit comprising a bisulfite(=disulfite, hydrogen sulfite) reagent as well as oligonucleotidesand/or PNA-oligomers according to claim
 22. 40. Use of a pretreatedgenomic DNA according to claim 22 for the characterisation,classification, diagnosis and differentiation of colon cellproliferative disorders.
 41. A kit comprising a bisulfite (=disulfite,hydrogen sulfite) reagent as well as oligonucleotides and/orPNA-oligomers according to claim
 26. 42. A DNA/PNA array for theanalysis of prostate cell proliferative disorders associated with themethylation state of genes comprising at least one nucleic acidaccording to claim
 26. 43. An array as recited in claim 42,characterized in that the solid phase surface is composed of silicon,glass, polystyrene, aluminium, steel, iron, copper, nickel, silver orgold.